【作者】 张林；

【导师】 巨容； 刘畅；

【作者基本信息】 电子科技大学 ， 儿科学， 2024， 硕士

【摘要】 新生儿坏死性小肠结肠炎(Necrotizing enterocolitis,NEC)是新生儿期常见的胃肠道危急重症,具有起病隐匿、进展迅速,在早产儿中的高患病率和高病死率等特点,有幸存活者往往遗留严重后遗症。然而其发病机制至今仍未完全阐述清楚,缺乏特异性治疗。研究进展认为肠道免疫稳态破坏和失调控的炎症级联反应是其重要病因,且有研究发现M2型巨噬细胞在抗炎调控和促进损伤愈合中发挥积极作用。因此,本研究旨在通过研究M2型巨噬细胞在炎性反应中的调控作用,进一步探索其来源的纳米囊泡对NEC的治疗作用,为NEC特异性生物治疗提供新的方案。囊泡制备:培养RAW264.7细胞系,分别使用LPS(终浓度100 ng/ml)和IL-4(终浓度100 ng/ml)刺激巨噬细胞极化为M1型和M2型巨噬细胞,将M2型巨噬细胞通过不同孔径(1μm、400 nm、200 nm)聚碳酸脂膜过滤后获得纳米级囊泡,高速离心获得囊泡液,通过透射电镜、纳米颗粒示踪分析进行囊泡形貌表征。疗效评价:(1)体外实验,将M2型巨噬细胞纳米囊泡(M2 nanovesicles,M2 NVs)与M1型巨噬细胞共培养48小时后,通过蛋白免疫印迹、酶联免疫吸附、流式细胞术和免疫荧光实验检测特征性因子。(2)体内实验:NEC模型鼠建立;将24只新生3天SD大鼠随机分配到对照组、NEC组、M2 NVs干预组,对照组新生鼠与母鼠同笼并维持纯母乳喂养,另两组新生鼠给予管喂配方奶每天4次+LPS(100ng/ml)0.2 ml 1次/日,M2 NVs干预组增加腹腔注射M2 NVs 0.3 ml(10~6个/ml)1次/日,第7日处死全部实验鼠;收集胃下端至直肠肠组织,据新生鼠坏死性小肠结肠炎模型肠道标本评分标准完成标本评估;苏木精-伊红染色后进行病理学观察;蛋白免疫印迹检测特征性因子;免疫荧光分析炎性环境改变。研究获得直径100-200 nm和颗粒密度在1.0-2.5×10~6个/ml的M2 NVs,呈类圆形,具有双层囊壁结构;体外实验发现,M2 NVs与M1型共培养后促进向M2型复极化,CD206、TGF-β和IL-4表达增高,免疫荧光中TNF-α表达减弱而TGF-β表达增强;体内实验发现,M2 NVs干预组肠组织损害更轻、肠道标本评分更低,促炎/抗炎性细胞因子表达降低/增高,免疫荧光发现M2 NVs干预组肠绒毛炎性微环境得到改善,肠损伤减轻。因此得出结论,M2 NVs能促进NEC模型肠组织中巨噬细胞由M1型向M2型复极化,改善NEC炎性环境中M1/M2比例,显著降低NEC肠组织中促炎性细胞因子表达,改善肠组织病理性损伤。更多还原

【Abstract】 Neonatal necrotizing enterocolitis(NEC)is a common critical illness of gastrointestinal tract in the neonatal period.It has some characteristics including hidden onset,rapid progression,high morbidity and mortality in premature infants,and often contributes to a serial serious sequelae.However,the pathogenesis has not been fully elucidated so far,and specific effective treatment has not been realized.At present,research progress of NEC pathogenesis suggested that the intestinal immune homeostasis disruption and the dysregulation of inflammatory cascade were important causes of NEC,meanwhile revealed that M2-type macrophages played a protective role in anti-inflammatory regulation and injury healing.Therefore,this study aims to investigate the regulatory role of M2-type macrophages in inflammatory response,further explore the therapeutic effect of nanovesicles derived from M2-type macrophages on NEC,and provide a new scheme for the specific biotherapy of NEC.Vesicles preparation:The RAW264.7 cell line was cultured,and macrophages were stimulated by LPS(final concentration 100 ng/ml)or IL-4(final concentration 100ng/ml)for acquirement of M1 or M2 macrophages.The M2 macrophages were filtered through polycarbonate membranes with different pore sizes(1μm,400 nm,200 nm)in sequence to obtain nanoscale vesicles,and the vesicles were purred through high-speed centrifugation.Then the vesicles were characterized by transmission electron microscopy and nanoparticle tracer analysis.Efficacy evaluation:(1)In vitro experiment,M2 NVs and M1 macrophages were co-cultured for 48 hours,and the expressions of related characteristic factors were detected by Western blot,Enzyme-linked immunosorbent assay,Flow cytometry and Immunofluorescence assay.(2)In vivo experiment:At first,NEC model was established and confirmed in neonatal SD rates.Secondly,Twenty-four neonatal 3-day-old SD rats were randomized to the control group,NEC group and M2 NVs intervention group.The neonatal rats in the control group were caged with dams and maintained exclusive breastfeeding,and the neonatal rats in the other two groups were fed formula 4 times a day and LPS(100ng/ml)0.2 ml once a day by gastric intubation,and the M2 NVs intervention group was added intraperitoneal injection of M2 NVs 0.3 ml(10~6/ml)once a day before execution.Finally,7 days after birth,all rats were executed,and intestinal tissues from the lower of the stomach to the rectum were collected.The intestinal specimens were evaluated according to the scoring criteria of neonatal rats necrotizing enterocolitis model,and pathological observation was performed after Hematoxylin-eosin staining.The expressions of characteristic factors were detected by Western blot,and the changes of inflammatory environment were analyzed by Immunofluorescence assay.Results show that M2 NVs with a diameter of 100-200 nm and a particle density of1.0-2.5×10~6/ml,double-layer vesicle wall structure,the shape of vesicles was like a circular or similar.In vitro experiments,M2 NVs co-cultured with M1-type macrophages could promote macrophages polarization toward to M2-type,and the increasing expressions of CD206,TGF-βand IL-4 were detected.Meanwhile,Immunofluorescence assay found the decreasing expression of TNF-αand the increasing expression of TGF-β.In vivo experiment,in the M2 NVs intervention group,the intestinal tissue pathological damages were milder,the intestinal sample scores were lower,the pro/anti-inflammatory cytokines expressions were decreased/increased respectively.Immunofluorescence showed that the intestinal villi inflammatory microenvironment was improved in the M2 NVs intervention group,and the intestinal injury was alleviated.Therefore,conclusions show that M2 NVs can promote the polarization of macrophages from M1 type to M2 type in the intestinal tissue of NEC model,improve the M1/M2 ratio in the inflammatory environment of NEC,significantly reduce the expression of pro-inflammatory cytokines in the intestinal tissue of NEC,and improve the pathological injury of intestinal tissue.更多还原